Insects and olfaction - General Introduction

General Introduction

1.4. Insects and olfaction

Insects belong to arguably the most successful and most diverse group of animals (Stork 2003). Out of 1.75 million species that have been formally described, 850,000 to 1,000,000 are insect species. However, the number of undescribed species is undoubtedly much higher. Insects comprise over half of the described species, and circa 3/4 of known animal species (Stork 2007). Whatever the global estimate, insects are highly diverse as illustrated in Figure 1-3. Not only insects are so abundant, but they have evolved to live on Earth for the last 400 million of years, with an extreme diversification and filling all available environmental niches (Grimaldi and Engel 2005). An outstanding feature is their sensory system. For instance, insect olfaction is highly evolved so that insects can search for food

Figure 1-3 This “speciescape” illustrates the relative diversity of insects in relation to other species groups. The relative diversity is proportional to the size of the organism and therefore in the illustration above the fly is much larger than all the other organisms (after Wheeler 1990 and Gullan and Cranston 1999).

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sources, shelters and mates. In highly social species, like the honey bee and ants, olfaction is used to recognize a huge variety of airborne molecules, providing the members of the colonies with a high sensory network (Forêt and Maleszka 2006). In some cases the perception of VOCs is so highly sensitive to reach values far below modern analytical equipments. One interesting example is the black jewel beetle, Melanophila acuminata (Coleoptera: Buprestidae) which can detect smoke odour as far as 50 kilometers to locate forest fires. Their larvae can develop only in burned wood (Schütz et al. 1999a). The most sensitive perception is evolved in the sex pheromone perception of male Lepidoptera.

Minute quantities of the so-called sex pheromones can attract males from huge distances to the female insects for mating (Kassiling 1979).

Figure 1-4 Scanning electron micrographs of the adult head of the rust red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). General overview of the insect ventral part showing the antennae, the mouth parts and the compound eyes. Bar: 100 µm. (with courtesy of Dr. Sergio Angeli).

-Insect sensory organs are housed in hair-like structures, known as sensilla, which are protruding from the cuticle of specific organs as antennae, mouth parts, and tarsal segments, but also on other parts of the insect body as wings and external genitalia as shown in Figure 1-4 and in Figure 1-5. The insect sensilla (singular = sensillum) protrude from the cuticle, or sometime lie within or beneath it. They can be divided in chemo-, mechano-, thermo-, visual and hygrosensory sensilla (Keil 1999). The structures of all sensilla types are rather uniform regardless of the specific receptor modality.

Figure 1-5 Schematic representation of the sensory organs and peripheral nerves of an adult fly. The main structural and functional subclasses of sensilla are represented in different colours. abn: abdominal nerves; bas: basiconic sensilla; cam: large campaniform sensilla of the wing blade; cns: central nervous system; iom: interommatidial bristles; mac:

macrochaetae; mic: microchaetae; wcs: chemoreceptors of wing margin; wms:

mechanoreceptors of wing margin (after Hartenstein 1993).

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Each sensillum consists of one or more bipolar receptor cells which send their axons to the brain and their dendrites to the peripheral cuticle region of stimulus uptake. A specific cuticular apparatus is present in the outside part of the sensillum and it exhibits features characteristic of the specific stimulus modality of the sensillum. Three types of auxiliary cells, thecogen, trichogen, and tormogen cells, surround the sensory neurones and border the sensillar lymph cavity (Figure 1-6). The sensillar cuticle plays an important role in stimulus transport to the receptor membrane of the sensory neurones. These neurones are surrounded by an extracellular fluid, the sensillar lymph, which composition is regulated by the auxiliary cells.

The chemosensory sensilla are divided in olfactory and gustatory sensilla. In insects the distinction between olfaction and taste is not as clear as in vertebrate, since olfactory sensilla can respond to substances in solution and gustatory sensilla can detect molecule in the vapour phase. However, the signal processing is however quite different for the two types of stimuli. The axons from all the olfactory neurones terminate in the antennal lobes, whereas the axons from gustatory sensilla terminate in the ganglion of the body segment to which the sensillum belongs, as for instance the suboesophageal ganglion for the sensilla of the maxillary palps. Insect olfaction is therefore defined as the neuronal transduction of all chemical compounds, which leads to the activation of antennal lobes, in analogy with the vertebrate where olfactory neurones terminate in the olfactory bulb (Schmuker and Schneider 2007).

Olfactory sensilla are more concentrated on the antenna and maxillary palps. Their cuticle structure shows the presence of numerous small wall pores, for this reason they are also called wall pore sensilla or multiporous sensilla.

The external morphology of these sensilla can be further distinct in sensilla trichodea, sensilla basiconica, and sensilla coeloconica. Sensilla trichodea have usually the external shape of hairs with a sharply pointed tip. They are usually the most abundant on the antennae.

Sensilla basiconica have generally a smooth surface and are covered with irregular dense wall pores detected in large number over the external cuticle. In some species of Lepidoptera it was found that long sensilla trichoidea respond to the female pheromone, while sensilla

-basiconica are tuned to the perception of plant odour also called green volatiles or general odours or (Steinbrecht et al. 1996; Tegoni et al. 2004) (Figure 1-6, sensillar type d).

Gustatory sensilla are also called contact chemoreceptors. Most of them are found on the month parts as labrum, maxillae and labium, but they also occur on the antenna, tarsi and even on the female ovipositor. In contrast to the olfactory sensilla, they lack wall pores but have a single, terminal pore, therefore they are also called uniporus or terminal pore sensilla. (Figure 1-6, sensillar type c).

The olfactory transduction of odorant stimuli is performed on the cell membrane of the sensory neurones. Odorants are first absorbed on the cuticular surface of the sensillum and are thought to reach the interior part via the wall pores. The cavity of the pore is in some cases connected with pore tubules which some times directly contact the sensory neurone membrane. The conversion of extracellular chemical signal to a neurone electrical stimulus is known as signal transduction. In insects before odorants are coded into electrical signals they interact with the sensillar lymph, while in vertebrate they interact with the nasal mucosa. The group of biochemical processes which take place between the sensillar wall pores and the dendritic membrane of the sensory neurones are known as

“perireceptor events” (Getchell et al. 1984). As a consequence, perireceptor events occur in the sensillar lymph which is an aqueous barrier, whereas odorants are often hydrophobic.

In the sensillar lymph odorants interact with different classes of soluble proteins:

odorant binding proteins (OBPs) (Vogt and Riddiford 1981), chemosensory proteins (CSPs) (Angeli et al. 1999) and odorant degrading enzymes (ODE) (Vogt and Riddiford, 1981). OBPs are present in very high concentration in the sensillar lymph and, similar to the CSPs, they reversibly bind chemical stimuli. Binding capacity was demonstrated with sex pheromones and general odorants for some members of insect OBP (Pelosi 1994, 1998; Steinbrecht 1998) and for one member of CSPs (Ban et al. 2003). OBPs and CSPs are soluble proteins with a low pI (4-5) and low molecular weight (10-14 kDa). Whether these proteins participate in odour coding or function as carrier to transport the odorants

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to the olfactory receptors of the sensory neurones it is still not clear although several hypotheses have been proposed (Pelosi 2005; Forêt and Maleszka 2006).

Figure 1-6 Schematic representation of insect sensilla (above). A: the cellular organisation; B and H: mechanosensory campaniform sensillum; C and G: gustatory or terminal pore sensillum; D, E and F: olfactory sensillum with single- (E) or double wall (F). ax:

axon of the receptor cell;

cu: cuticle: de: dendrite ep:

epidermal cell (yellow);

ne: receptor neurone (red);

th: thecogen cell (green);

to: tormogen cell (light brown); tr: trichogen cell (dark brown); sl: sensillar lymph (blue). Schematic representation of the insect olfactory transduction (under). (after Steinbrecht 1992).

-Odorant receptors are a group of transmembrane proteins, belonging to the class of G-protein coupled receptors. Once the odorant receptors are activated they drive two alternative intracellular signalling pathways, one utilising cAMP and the other inositol triphosphate (IP3) as second messengers (Raming et al. 1993). These second messengers travel across the cell cytoplasm and activate gated ion channels, allowing Ca⁺⁺ (or other cations) to flow inside the cell. The increase in intracellular Ca⁺⁺ concentration appears to activate chloride a chloride current that helps to depolarise the olfactory cell leading to the generation of an electrical signal or action potential (Krieger et al. 1997).

1.5. Electroantennography (EAG)

Electroantennography is a technique to measure the electrical activity generated by an antenna for a given odorant. It is commonly used to study the function of the olfactory system in insects. The technique was developed after the discovery by the German biologist Dietrich Schneider (1957), who measured voltage changed between the tip and base of a freshly excised antenna from a male of the silkmoth, Bombyx mori, while the antenna was stimulated with an air puff containing the silkmoth sex pheromone bombykol. Schneider named this odour-prompted electrical response of an insect antenna an “electroantennogram” (EAG). It is interesting to note that his idea started thanks to a meeting with Schneider’s neighbour (P. Karlson), who provided Schneider bombykol, the first discovered pheromone of animals (Butenandt et al. 1959) as nicely described later by Schneider (1999).

The EAG response is a bulk measure of the responses of the electrical depolarisations of many olfactory receptor neurones cells when the insect antenna is exposed to adequate stimulus (Figure 1-7). EAG responses are therefore related to the total number of stimulated sensilla (Mayer et al. 1984), although only in the recent years an explicit relationship to the neuronal activities has been demonstrated. The numbers of spikes elicited from receptor cells and the change in the EAG potential are interdependent measures of the stimulus strength (Mayer 2001).

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Figure 1-7 Electroantennography. (A) The antenna is exposed to stimulus molecules and the voltage changed between recording and reference electrodes is registered, after the signal is amplified and processed. (B) A typical antennal responses of the female beetle Cis boleti to different compounds at 10^-3 paraffin dilution, and (C) dose response curve of C.

boleti to different dilutions (form 10^-6 to 10^-3) of 1-octen-3-ol.

C

1-octen-3-ol

A

B

-EAG registrations can be done in two ways: by exciting the antenna from the insect and make it in contact with two electrodes or by inserting a ground wire in an immobilised insect and another wire to the tip of its antenna (Figure 1-7). The latter method has the advantage that the insect antenna remains alive for longer time, therefore EAG signals can last very long. On the other hand, in this case the measurement is often disturbed by higher signal noise, due to the mechanical movement of the antenna and/or of the full animal.

The amplitude of the EAG responses is influenced many factors:

• nature and strength (concentration) of the stimulus;

• condition of the antenna e.g. sex, physiological status and previous stimulations;

• operating conditions e.g. temperature and humidity;

• quality of the amplifier input.

The insect circadian rhythm may also influence olfactory response, as it has demonstrated in the fruitfly, Drosophila melanogaster (Krishnan et al. 1999), and in the evolutionarily distant cockroach, Leucophaea maderae (Page and Koelling 2003). In the first case, EAG responses of flies tested near the middle of the night were significantly higher than those of flies tested at other times of the day. In the second case, a ten fold variation of the EAG amplitude was observed by testing odorants as specific time of the circadian rhythm. These experiments suggested that olfactory responses and circadian rhythm are linked with the photoreception and with the neuronal signalling of the insect optic lobes.

EAG technique has been further integrated with gas chromatography, by attaching an EAG detector to a gas chromatograph. This powerful analytical technique is known as a GC-EAD (gas chromatography-electroantennographic detection). With his apparatus it is possible to explore how insect antennae respond to different chemical compounds released by a given source as host plants or food substrates. To identify the volatile compounds detected by insects it is in this case necessary to run the samples separate in GC-EAD and GC-MS. This problem has been recently solved by the integration of the two systems. In this

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case the volatiles samples are injected in a gas chromatograph - mass spectrometer/electroantennographic detection (GC-MS/EAD), where the volatile substances are spitted between a mass spectrometer and a EAD detector at the end of the GC column (Figure 1-2) (Weissbecker et al. 2004). This system proved to be highly sensitive to compounds of importance to insects (able to detect them in the sub-picogram range) and profitably assists in chemical ecology research.

1.6. Biosensors

A biosensor is defined by the International Union of Pure and Applied Chemistry (IUPAC) as a “device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals” (Rasooly 2005). A biosensor consists of two main parts: a biological component also called biocomponent and a signal- transducing component. The biocomponent is a living organism or a biological product.

The schematic of biosensor with some accessories is depicted in Figure 1-8.

Figure 1-8 Schematic representation of a biosensor. A biosensor consists of two main parts: a biological component also called biocomponent (leftmost), which is sensitive to a target chemical compound (A) and a signal-transducing component. The latter is here represented by a transducer, an amplifier, a processor and a display. The biosensor may be equipped with a calibration part, here represented by a reference.

-Biosensor devices have great prospects in commercial applications in such fields as biomedicine, environmental monitoring, pharmacology, agriculture, quality control of food and water, and military applications. Biosensors are now being developed for detection of microbial pathogens and their toxins, monitoring and analysing blood metabolites, cancer monitoring and detection, allergen detection, food and biomaterial quality testing, and basic research on molecular interactions.

The most widespread example of a commercial biosensor is perhaps the blood glucose biosensor, which measure the concentration of blood glucose by using an enzyme to break down glucose. In doing so there is a transfer of electrons to an electrode, allowing a measurement of blood glucose concentration (Fogh-Andersen and D'Orazio 1998). Other examples are the use of yeast and filamentous fungi as sensing elements for detecting cell activities (Boranian 2004), the use of anaerobic bacteria as biosensors for analysing degradable organic matter in waste water (Kumlanghan et al. 2007), and the use of specific enzymes in amperometric biosensors for food analysis (Prodromidis and Karayannis 2002). Moreover, biosensors are developed in military applications as to detect buried explosives by using bacteria as sensing organisms (US Patent 5972638).

An ideal biosensor offer several advantages over other analytical methods including rapid and even real-time measurements, high sensitivity, selectivity, and stability (a rule known as “the three-S-rule”) even when a complex or turbid sample matrix is used.

Olfactory-based biosensors have been fabricated by combining olfactory biocomponents with various signal-transducing devices. Olfactory biocomponents are for instance: intact insects, insect antennae, olfactory receptor neurons, and olfactory receptor proteins. These biocomponents are combined with detecting devices such as quartz crystal microbalance (QCM), field effect transistor (FET), microelectrode, surface plasmon resonance (SPR) and light addressable potentiometric sensor (LAPS) (Wu et al. 2007).

In this thesis we refer to the term “antennosensor” as an olfactory-based biosensor where the biocomponent part is an insect antenna, as the name hints. Insect antennae are highly sensitive to selective volatile compounds, wherefore they are ideal biocomponents of an olfactory-based biosensor. The study of insect olfaction started since the discovery

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of the first sex pheromone of the silkmoth, Bombyx mori (Lepidoptera: Bombycidae) as previously described (Schneider and Hecker 1956). A review about these aspects has been written by Keil (1999). Male moth antennae are extremely sensitive to their female sex pheromones. For example, amounts of less than 10 pg of bombykol offered in a piece of filter paper elicit a typical behavioural response in the males (Kassiling 1979). Another extreme case was found in a jewel beetle Melanophila acuminata (Coleoptera: Buprestidae) which could detect guaiacol derivatives produced from forest fire in a range of part per billion (ppb) and in a distance as far as 50 kilometres (Schütz et al. 1999a).

The advantage of high volatile recognition allow insect antenna to be exploited as a biocomponent in a biosensor, providing fast and non-destructive detections. There are several applications by using insect antennae as detectors, especially in agriculture. For example, the antennae of Colorado potato beetle Leptinotarsa decemlineata (Coleoptera:

Chrysomelidae) were used for identifying fungal-infested potatoes (Schütz et al. 1999b).

By detecting the odorant 2-ethyl-hexane-1-ol, one single infested potato could be found up to 100 kg of healthy potatoes (Schütz et al. 1999b).

In other cases the antennae of Pectinophora gossypiella (Lepidoptera: Gelechiidae) and Cydia pomonella (Lepidoptera: Tortricidae) were used as biocomponents of portable biosensors to detect the ambient distribution of species specific pheromones in cotton fields (Färbert et al. 1997; Koch et al. 2002) and apple orchards (Koch et al. 1997), respectively. These applications are of high values in biocontrol of pest insects, where pheromone dispensers are adopted of mating disruption approach (Angeli et al. 2007).

-Figure 1-9 Schematic drawing of the biosensor-system. (A) Sampling and calibration system. (B) Antenna holder. 1: antenna, 2: reservoir of electrolyte, 3: Ag/AgCl-electrode.

(C) Recording of 1 measurement cycle using superposition technique in a lepidopteran species. Upper trace: EAG recording. Lower trace: recording of syringe and filter activity.

Vertical bars indicate syringe concentration of the puffs. K1, K2, K3: EAG amplitude generated by increasing odorant concentration when filter air was in the background. Z1, Z2, Z3: EAG amplitude generated by increasing odorant concentration when ambient air was the background. (D) Recording of 1 measurement cycle using superposition technique in a coleopteran species. First 4 EAG amplitudes (from left) generated when filter air was in the background. Second 4 EAG amplitudes (right) generated when ambient air was in the background. Draw A, B and D after Schütz et al. 1999b; draw C after Färbert et al. 1997.

A

C D

B

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Olfactory-based biosensors can be grouped in two categories: qualitative biosensors, which provide information only in respect to the presence or not of the target compounds, and quantitative biosensors, which allow quantifying the concentration of the target compounds. A biosensor of the first type has been developed by van der Pers and co-workers (van der Pers and Minks 1998).

In case of quantitative biosensors have to hold a calibration part. In other words, the electrical signal, detected from the biocomponent and recorded in the transducing component, has to provide information about the concentration of the chemical stimulant. To achieve this goal it is important to isolate the background detection, which affects the target compound detection, and to calibrate the system.

In the antenna-based biosensor utilised in the present thesis, these parameters were controlled as described fully by Färbert et al. (1997). Since this is a critical part of the antenna-base biosensor a briefly description is here provided. The background effect was controlled by exposing the antenna first to a filtered and clean air and subsequently to the ambient air, where a target compound had to be measured. Antennal responses were measured by air puffs from three syringes previously equipped with defined concentrations of the target compound.

The concentration of the target compound in the ambient air was achieved by measuring first the antennal responses to define concentrations of the target compound when the antenna was exposed to filtered air, and then similar measurements were repeated when the antenna was exposed to the ambient air. The first three antennal responses were therefore not influenced by the ambient air, while the last three antennal responses are measured as extra signals, meanwhile the antenna was affected by the

Im Dokument Development of a Basic Biosensor System for Wood Degradation using Volatile Organic Compounds (Seite 34-55)